Centrifugal device and method for performing binding assays

ABSTRACT

The invention relates to a device for performing binding assays. In particular, the invention relates to a centrifugal device for performing such assays. The invention also relates to a method of performing binding assays involving antigen-antibody binding, nucleic acid hybridization, or receptor-ligand interaction.

TECHNICAL FIELD

The invention the subject of this application relates to a device forperforming binding assays. In particular, the invention relate to acentrifugal device for performing such assays. The invention alsorelates to a method of performing binding assays involvingantigen-antibody binding, nucleic acid hybridization, or receptor-ligandinteraction.

BACKGROUND ART

A fundamental aspect of research in the biological and medical sciencesis the measurement of the binding of one chemical entity to anotherchemical entity. Such measurements are usually referred to as bindingassays and include the measurement of the binding of an antigen to anantibody or vice versa, the bending of one nucleic acid to anothernucleic acid such as in a hybridization reaction, and the binding of aligand such as a hormone or other effecter molecule to its receptor.

There are numerous techniques available for performing binding assayswith the technique employed for a particular assay usually beingdictated by the types of molecules involved in the interaction. Ingeneral, however, one of the partners in the interaction is bound to asolid support such as a membrane or the walls of wells in microtitreplates. Many of the known techniques are automated and are adapted forthe simultaneous assaying of multiple samples.

While known techniques permit the efficient performance of numerousassays in a given period, the techniques have limitations. The mostserious limitation is that a particular technique and the apparatusassociated therewith can usually only be used for a single bindingassay. Many of the techniques further suffer from the complication thatmultiple steps are involved in which reagents have to be sequentiallyadded and removed.

It would therefore be desirable to have available apparatus that can beused for performing a variety of binding assays—evensimultaneously—under variable conditions and by which the assays can bedone with avoidance of the multiplicity of steps necessary in knownprocedures.

The object of the invention is to provide such an apparatus and methodsutilizing that apparatus.

SUMMARY OF THE INVENTION

In a first embodiment of the invention, there is provided a device formeasuring the binding of a first partner in an interaction to a secondpartner in said interaction, said device comprising:

-   a) an opaque temperature-controlled chamber having a rotor therein,    said rotor having at or near the periphery thereof at least one    radially positioned transparent reaction well, said reaction well    having on an upper surface thereof an aperture for the addition of    reagents thereto, said reaction well further including on an    internal surface thereof at the end closest the axis of said rotor    at least one attachment zone for said second interaction partner;-   b) a system for detecting light emitted or absorbed by said first    interaction partner or an indicator molecule bound thereto; and-   c) means for controlling the temperature of said chamber and the    operation of said rotor.

In a second embodiment, the invention provides a method of measuring thebinding of a first partner in an interaction to a second partner in saidinteraction, said method comprising the steps of:

-   a) delivering a quantity of second interaction partner to a reaction    well of a device according to the first embodiment for attachment of    said second interaction partner to an attachment zone of said    reaction well;-   b) combining a quantity of first interaction partner with said    second interaction partner in said reaction well and incubating said    mixture at a temperature and for a time to allow binding of said    first interaction partner to said second interaction partner;-   c) rotating said device rotor at a speed which displaces the mixture    formed in step (b) away from said attachment zone; and-   d) measuring the amount of said first interaction partner bound to    said second interaction partner via the fluorescence or absorbance    of said first interaction partner or an indicator molecule bound    thereto.

Other embodiments of the invention will become apparent from a readingof the detailed description below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a semi schematic representation of a rotor of a deviceaccording to the invention with detail of a optical detection systemincluded.

FIG. 2 is a representation of one of the reaction wells of a rotor of adevice according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The term “interaction” is used herein to denote the binding of anymolecule (the first interaction partner) to another molecule (the secondinteraction partner) where the interaction may be a naturally occurringinteraction or the binding of a synthetic molecule to a target molecule.

The interaction partners can be any pair of molecules in which a firstmolecule can bind to the second molecule. The term interaction partnertherefore includes, but is not limited to, the following pairs ofmolecules: First Interaction Partner Second Interaction Partner AntibodyAntigen Antigen Antibody Enzyme Substrate Oligopeptide Protein (forexample, an enzyme or receptor) Hormone Receptor Effector moleculeReceptor Nucleic Acid (RNA or DNA) Nucleic acid (RNA or DNA)Oligonucleotide Nucleic acid (RNA or DNA) Synthetic organic compoundProtein (for example, an enzyme or receptor)

The terms “comprise” and variants of the term such as “comprises” or“comprising” are used herein to denote the inclusion of a stated integeror stated integers but not to exclude any other integer or any otherintegers, unless in the context or usage an exclusive interpretation ofthe term is required.

With regard to the first embodiment of the invention as define above,the device chamber can be any suitable, typically insulated, containerfor the rotor and other device components. The chamber advantageouslyhas a lid or sealable opening to allow loading of reaction wells. Thechamber must be opaque—that is, impermeable to light—to allow accurateand sensitive measurement of the second interaction partner molecule orindicator molecule.

The temperature control of the device chamber is effected by providing aheater linked to a temperature sensor so that a set temperature can bemaintained. Typically, heating is by a heater located within the chamberwith circulation of heated air within the chamber aided by a fan.Alternatively, heated air can be supplied to the chamber from a port orports in a chamber wall. Heating of the chamber can also be by infraredradiation.

Temperature control can also include a cooling system. For example, airsupply to the chamber can be provided wherein the air is either atambient temperature or less than ambient by passage through or over acooling means. The temperature sensor referred to above isadvantageously linked to the cooling system.

The device rotor is typically a flat disc of a plastic or metal materialhaving reaction wells fitted therein. The reaction wells can beremovable or the entire rotor with reaction wells can be a disposableitem. The rotor of devices according to the invention advantageouslycomprise a plurality of reaction wells. The number of wells will dependon the configuration of the device but a typical range is 1 to 96 wells.

Reaction wells can be formed from any suitable transparent material suchas polypropelene or polycarbonate. The wells can be cylindrical,rectangular prisms, or any other suitable shape provided that the wellis of sufficient length to provide an area for the at least oneattachment zone and an area into which the solution or solutions viawhich the interaction partners were applied can be displaced bycentrifugal force.

In some embodiments, the reaction wells can be angled upwards toward theperiphery of the rotor. This allow solution to migrate back to the atleast one attachment zone once rotor speed has been sufficientlyreduced. The function of such migration will be explained below.Transitory vibration of the rotor can also be used to effect migrationof solution in a reaction well and in such instances the well does nothave to be tilted and can be horizontal.

Attachment zones are typically provided by appropriately treating thesurface, spotting the second interaction partner onto the attachmentzone with a pin or ink-jet, and drying the spotted component.Alternatively, a magnet can be provided beneath the desired area of areaction well. The second interaction partner is linked to a magneticparticle which is held in the attachment zone by the magnet. Attachmentzones typically have a diameter, if a circular zone, of 50 μm to 3 mm. Aparticular reaction well can have a plurality of attachment zonesallowing different second interaction partners to be delivered to thatwell if desired.

The rotor drive means can be any drive means used for rotor devices inscientific equipment. For example, the drive means can be direct-coupledAC motor, a DC motor, or an AC motor that drives the rotor via a gearboxor pulleys or the like. Preferably, the drive means is a direct-coupledAC motor, DC motor or stepper motor with the motor external to thechamber.

The detection system comprises a light source and a detector. Thesecomponents can be any of the light sources and detectors know to thoseof skill in the art. For example, the light source can be an LED, alaser light source or a halogen lamp, with an appropriate filter toprovide light of an appropriate wavelength for:

-   a) excitation of any fluorophore associated with the first    interaction partner (by associated it is meant that the fluorophore    is directly linked to the first interaction partner molecule or is    linked to an indicator molecule); or-   b) absorbance by the first interaction partner or indicator    molecule.

The detector will be suitable for the measurement of emitted fluroesenceor absorbance. Detection systems advantageously include both types ofdetectors to give the device greater versatility in the types of bindingassays that can be performed.

A device according to the invention can have associated therewith acomputer for controlling such operations as:

-   Rotor speed;-   Chamber temperature;-   Time and temperature for annealing and polymerization steps when the    binding assay is an hybridization;-   Rotor braking;-   Vibration of the rotor; and-   Processing of data generated by the detection system.

A device that can be suitably adapted for use in the present inventionis that described in International Application No. PCT/AU98/00277(Publication No. WO 98/49340) the entire content of which isincorporated herein by cross reference.

With regard to the second embodiment of the invention, a method ofperforming a binding assay utilizing the device of the first embodiment,the quantities of first and second interaction partners areadvantageously delivered as solutions which can contain other componentssuch as buffers, salts, DNA or RNA polymerization reagents including apolymerase, or a blocking reagent if necessary. Solutions of interactionpartners can be delivered by any of the methods known to those of skillin the art.

Temperatures and incubation times will be in accordance with theparticular binding assay being performed and those parameters will beknown to those of skill in the art. For example, hybridization reactionscan be performed as described in Molecular Cloning: A Laboratory Manual,Second Edition (J. Sambrook et al., ed's), Cold Spring HarbourLaboratory Press, 1989. For performing a polymerase chain reaction,typical reaction mixtures and conditions are described, for example, instandard texts such as PCR: a Practical Approach (M. J. McPherson etal., ed's), IRL Press, Oxford, England, 1991, and numerous brochuresprovided by suppliers of amplification reagents and consumables. Theentire content of the foregoing publications is incorporated herein bycross reference.

Binding of the first interaction partner to the second interactionpartner can be done with the rotor spinning. The solution containing themixture of interaction partners can usually be spun at a speed of lessthan 500 rpm while retaining the solution at the attachment zone. Aspeed of greater than 500 rpm is usually sufficient to displacesolution—and hence any unbound first binding partner—away from theattachment zone. The bound first interaction partner is then measured byway of the detection system.

To allow measurement of bound first interaction partner, the partnerhas:

-   An inherent absorbance (which is different, if necessary, at the    selected wavelength to the absorbance of other components of the    binding assay); or-   Has linked thereto a fluorescent or absorbent group.

Suitable fluorescent and absorbent groups will be known to those ofskill in the art. Typical fluorophores include those abbreviated as FAM,JOE, ROX, TAMRA, Cy5, Cy3, Cy5.5, and VIC. Typical absorbent groups areDabcyl and BH quenchers.

Alternatively, the bound first interaction partner can be measured byallowing an indicator molecule to bind thereto. This can be done as afurther step after unbound first interaction partner is displaced formthe attachment zone. Excess indicator molecule is similarly removed bycentrifugal force after a sufficient period has been allowed at asuitable temperature for binding of the indicator molecule to the boundfirst interaction partner.

In instances where the first and second interaction partners form a DNAduplex or a DNA duplex is formed as a PCR product, an intercalating dyecan be used to detect the duplex. Such dyes will be known to those ofskill in the art. A particularly preferred dye is Sybr green.

The indicator molecule can be any molecule that is fluorescent orabsorbs at an appropriate wavelength and which binds to the firstinteraction partner. Typically, the indicator molecule is anappropriately derivatised antibody that is specific for the firstinteraction partner.

Absorbance or fluorescence is measured with the rotor spinning,typically at a speed of at least 500 rpm at which speed the mixture willmove away from the attachment zone. Data capture is controlled so thateach attachment zone that passes over or under the detector isindependently measured. Multiple detectors can be provided for use withreaction wells that include multiple attachment zones. Alternatively, asingle detector can be used, with appropriate control, to scan allattachment zones.

A device according to the invention will now be described with referenceto the accompanying figures. FIG. 1 shows rotor 1 of a device having aplurality of reaction wells mounted thereto, one of which wells is item2. A laser or diode 3 is provided as a light source, light from which isdirected through a beam splitter or dichroic mirror 4. Emittedfluorescence or absorbed light is measured by detector 5.

FIG. 2 shows a reaction well 6 which in this instance is a rectangularprism fabricated from polypropylene. Well 6 has at its end which isclosest the axis of the rotor when the well is in situ a plurality ofattachment zones one of which is item 7. The upper surface of well 6 hasa loading port 8. In use, after reagents have been added to the well,the solution thereof is displaced to end 9 of the well by centrifugalforce through rotation of the rotor holding well 6. Bound firstinteraction partner is then detected as schematically represented byarrow 10.

Examples of binding assays that can be performed using the device andmethod of the invention will now be given.

Realtime Detection of PCR Products

A probe or a primer is bound at the attachment site, and PCR performedin the vessel. The following are done during PCR cycling:

-   1) After each cycle of PCR at high rotor speed (greater than 500    rpm) the rotor is slowed so that the reaction mix covers the    attachment zone (this is done at the annealing temperature of the    primer/probe to the PCR product).-   2) After a specified annealing time, the rotor speed is increased to    remove the PCR mix from the attachment zone.-   3) Fluorescence readings are taken at the attachment zone.-   4) The previous steps are repeated.    Determination of Probe Melting Temperature

A primer or probe is bound to an attachment zone of a reaction well of adevice according to the invention. A PCR reaction mix is added to thevessel, that includes at least one fluorescently-labeled primer. Therotor is spun at high speed to ensure no contact with the attachmentzone and then the vessel cycled to perform PCR amplification. Afteramplification, the rotor is slowed to allow the PCR mix to come intocontact with the attachment zone, so that the PCR product will hybridizespecifically to the immobilized probe or primer. The temperature isreduced to below the expected melt temperature during this step to allowhybridization to occur. The rotor is then spun at high speed, thefluorescent signal at the attachment zone is sampled and the temperatureof the chamber increased slowly, typically at 0.20° C. per second. Thefluorescent signal captured will confirm the melt temperature of theprimer/probe and PCR product.

Reference was made above to vibration of the rotor to move solution backover the attachment zone. This can also be achieved through use of atilted reaction well. On sufficient slowing of the rotor, solution movesfrom an upward position at the distal end (relative to the axis of therotor) to a lower portion over the attachment zone area of the reactionwell. Rotor vibration is thus not needed in this embodiment of thedevice.

Determination of Antigen Levels in a Sample

An antibody specific for the antigen of interest is bound at anattachment zone of a reaction well. A sample containing an unknownamount of antigen is then allowed to react with the antibody. Solvent isremoved by centrifugation and a solution containing an antibody to theantigen is applied to the attachment zone. This antibody may be the sameor different to the antibody referred to above but is fluorescentlylabeled. Excess second antibody is then removed by centrifugation andthe amount of antigen measured by fluorescence.

SNP Detection Using PCR

Synthetic probes for the mutated and wild type sequences are bound toseparate attachment zones of a reaction well. The well is then loadedwith a reaction mixture containing a DNA sample and a PCR master mix.The forward primer is labeled with a fluorophore. After each PCR cycleat the annealing temperature, the rotor speed is reduced and thereaction mixture allowed to overlay the immobolised probes. The rotorspeed is then increased to displace reaction mixture and thefluorescence of the DNA retained at the attachment zones measured. Anincrease in fluorescence indicates amplification of either mutant,wild-type, or both DNAs.

Alternatively, a forward primer that is complementary to a portion ofthe mutated DNA is labeled with a fluorophore, for example FAM. Aforward primer that is complementary to a portion of the wild-typesequence is labeled with a different fluorophore, JOE for example. Aprobe that is homologous to both the mutation and wild-type PCR productsis bound at an attachment zone of a reaction well. By measuring the JOEto FAM ratio of PCR product hybridized with the probe the genotype canbe determined.

It will be appreciated that many changes can be made to the device andmethod of use as exemplified above without departing from the broadambit and scope of the invention.

1. A device for measuring the binding of a first partner in aninteraction to a second partner in said interaction, said devicecomprising: a) an opaque temperature-controlled chamber having a rotortherein, said rotor having at or near the periphery thereof at least oneradially positioned transparent reaction well, said reaction well havingon an upper surface thereof an aperture for the addition of reagentsthereto, said reaction well further including on an internal surfacethereof at the end closest the axis of said rotor at least oneattachment zone for said second interaction partner; b) a system fordetecting light emitted or absorbed by said first interaction partner oran indicator molecule bound thereto; and c) means for controlling thetemperature of said chamber and the operation of said rotor.
 2. A methodof measuring the binding of a first partner in an interaction to asecond partner in said interaction, said method comprising the steps of:a) delivering a quantity of second interaction partner to a reactionwell of a device according to the first embodiment for attachment ofsaid second interaction partner to an attachment zone of said reactionwell; b) combining a quantity of first interaction partner with saidsecond interaction partner in said reaction well and incubating saidmixture at a temperature and for a time to allow binding of said firstinteraction partner to said second interaction partner; c) rotating saiddevice rotor at a speed which displaces the mixture formed in step (b)away from said attachment zone; and d) measuring the amount of saidfirst interaction partner bound to said second interaction partner viathe fluorescence or absorbance of said first interaction partner or anindicator molecule bound thereto.